The quantitative separation of chylomicrons and chylomicron remnants by column chromatography

93

Biochimica et Biophysics Acta 918 (1987) 93-96 Elsevier

BBA Report

BBA 50173

The quantitative

separation of chylomicrons

and chylomicron remnants by column chromatography James M. Felts Lipid Research Lob, Veterans Administration Medical Center, San Francisco, CA (U.S.A.) and Department of Physiology, University of California, San Francisco, CA (U.S.A.) (Received 26 November 1986)

Key words: Chylomicron; Chylomicron remnants; Lipoprotein; Free fatty acid

Chylomicrons and chylomicron remnants cannot be separated by classical techniques of uhracentrifugation or gel filtration, since there is a marked overlap of density and size. Chylomicron remnants develop a high negative surface charge during their formation presumably due to surface-oriented free fatty acids. Two chromatographic matrices have been identified which separate these two lipoprotein species based on the charge differences. Both DEAESephacel and protamine-Affi-Gel 10 effect a quantitative separation. These techniques may be useful in studies of chylomicron metabolism both in vivo and in vitro.

Chylomicrons have a dominant role in the transport of triacylglycerols and cholesterol of dietary origin [ 11. The initial step in the metabolism of these triacylglycerol-rich lipoproteins is the interaction with lipoproteins lipase (EC 3.1.1.34) situated on endothelial surfaces of extrahepatic tissues [2]. Evidence has been presented that chylomicrons are not directly removed by the liver [3]. Hydrolysis of a fraction of the triacylglycerol of chylomicrons by lipoprotein lipase in peripheral tissues produces smaller, modified particles or chylomicron ‘remnants’ which are subsequently removed by the liver [4-61. The ability of the liver to ‘recognize’ and metabolize remnants, but not chylomicrons, might be determined by differences in chemical composition or physical properties of these two lipoproteins. Changes in lipid composition, apolipoprotein composition and size during remnant formation have been reported [4,5,7]. Remnants contain less triacylglycerol and more Correspondence: J.M. Felts, Chief, Lipid Research (151L), VA Medical Center, 4150 Clement Street, San Francisco, CA 94121, U.S.A. 0005-2760/87/%03.50

cholesterol, cholesterol esters, diacylglycerols, monoacylglycerols and have more apolipoprotein E than chylomicrons. During the course of formation of remnants by the interaction of chylomicrons with lipoprotein lipase, additional components are added to the surface. We have shown that remnants produced in vivo acquire lipoprotein lipase undoubtedly derived from endothelial surfaces [6]. Remnants produced in vitro from chylomicrons and post-heparin serum also contain associated lipoprotein lipase [6]. We have also noted that remnants have a markedly increased surface negativity compared to chylomicrons. This is probably due to the marked increase in the free fatty acids of the particle produced by triacylglycerol hydrolysis. It would be expected that the generated free fatty acids, due to their amphiphilic nature, would reside primarily at the lipoprotein surface [8]. The metabolisms of chylomicrons and remnants have been difficult to study, since they overlap with respect to particle size and density. We have taken advantage of the marked increase in surface negativity of the remnants to devise two

0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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chromatographic techniques which completely separate these two lipoprotein species. Male Simonsen rats (220-350 g) were maintained on Simonsen rat chow (Simonsen Laboratories, Inc., Gilroy, CA). Chylomicrons were obtained from a thoracic duct fistula by a modification of the technique of Bollman et al. [9]. Diethyl ether anesthesia was used. Rats were intubated 2 h prior to surgery with 3 ml of an emulsion of milk and corn oil (2 : 1, v/v) dispersed with a Polytron homogenizer. Some preparations were labeled with 0.05 mCi of [9,10-3H]oleic acid or [1-‘4C]oleic acid (NEN Research Products, Boston, MA) by coating the glass tube with the labeled fatty acid before dispersion of the corn oil mixture. Chyle was collected and chylomicrons were isolated by layering the chyle under 2-4 ml of 0.15 M NaCl in an ultracentrifuge tube and floated in an SW-40 rotor (Beckman Instruments, Inc., Palo Alto, CA) at 5 . lo6 g. min at 5°C in an L2-65B ultracentrifuge. The chylomicron pellet was isolated with a tube slicer and was resuspended in 0.15 M NaCl and made up to the original volume of chyle (20-30 mg triacylglycerol/ml). Remnants were produced in vivo by the injection of the isolated chylomicrons (20-40 mg triacylglycerol) into the jugular vein of a supradiaphragmatic rat preparation [lo] maintained under Nembutal anesthesia. This is a rapid method of obtaining a hepatectomized rat preparation. After 25 min the preparation was exsanguinated via a carotid artery cannula while injecting 5-10 ml of 0.15 M NaCl into the jugular vein. The diluted blood was allowed to clot and the serum was removed. The diluted serum was transferred to an ultracentrifuge tube and overlayed with 0.15 M NaCl. Remnants were floated in an SW-40 rotor at 10.106 gemin at 5OC in an L2-65B ultracentrifuge. The top fraction (1 ml) was isolated with the aid of a tube slicer and was resuspended in 0.15 M NaCl. For the production of remnants the circulation time of chylomicrons in the supradiaphragmatic rat is critical. If bleeding is carried out earlier than 25 min, variable amounts of unreacted chylomicrons are still in the plasma, thereby yielding a mixture of chylomicrons and remnants in the isolated lipoprotein fraction. Triacylglycerols were determined by the technique of Kessler and Lederer [ll] and Fletcher

[12]. Elution profiles were monitored by lightscattering of appropriately diluted fractions at 280 nm or at 700 nm or by assaying aliquots of each fraction for radioactivity. Aliquots were counted in 15 ml Scintiverse (Fisher Scientific, Inc., Fairlawn, NJ) in a liquid scintillation spectrometer (Packard Instrument Co., Downers Grove, IL) set for either 3H or 14C. Columns for the chromatographic separation of chylomicrons from remnants were prepared as follows. DEAE-Sephacel columns: DEAE-Sephacel (Pharmacia, Inc., Piscataway, NJ) was added as a dilute slurry to polypropylene disposable columns (Bio-Rad Econocolumns, Bio-Rad, Richmond, CA) and were packed to the 1.8 ml level. The Sephacel was washed with 10 column volumes of 0.15 M NaCl, 0.02 M Tris, pH 8.6. The lipoprotein mixture to be chromatographed was applied in 200 ~1 and elution was carried out with an NaCl gradient from 0.15 M to 1 M, 0.02 M Tris. Flow rate was set at 4 drops/mm and fractions of 0.75 ml were collected. The elution profile was determined as stated above. Protamine-Affi-Gel-IO columns: 10 ml of Affi-Gel10 (Bio-Rad) gel slurry was washed three times with 20 ml deionized water and the gel cake was mixed with 7.5 ml of 0.1 M NaHCO, (pH 8.3)’ containing 300 mg protamine sulfate (Grade X, Sigma Chemical Co., St. Louis, MO). This mixture was incubated at room temperature with gentle shaking for 1 h. To this was added 0.5 ml of 1 M ethanolamine (Sigma) and the mixture was allowed to stand overnight. Portions of the gel were packed in disposable columns to the 1.8 ml level and washed with 50 ml 0.1 M NaHCO, followed by 8 ml 6 M urea. The column was then equilibrated with the elution buffer (0.15 M NaCl, 0.02 M Tris, pH 8.6). The lipoprotein mixture to be chromatographed was applied in 200 ~1 and elution was carried out with an NaCl gradient from 0.15 M to 2 M, 0.02 M Tris. The elution profile was determined as stated above. The following column packing materials were ineffective in separating chylomicrons from remnants: immobilized protamine (Pierce Chemical Co., Rockford, IL), Cellulose (Sigmacell, Sigma), DEAE-cellulose (Cellex D, Bio-Rad), TEAE-cellulose (Cellex T, Bio-Rad) and hydroxyapatite

95

(Bio-Gel HTP, Bio-Rad). On most of these materials remnants adhered, but could not be eluted even with 5 M NaCl. It has previously not been possible to separate chylomicrons from remnants by classical techniques of ultracentrifugation or gel filtration due to the fact that the two lipoproteins overlap with respect to density and size [7]. Thus, previously remnants have been defined by an operational definition as partially metabolized chylomicrons which are recognized by the liver. We report here a marked alteration in surface charge on remnants which gives them unique physi~he~cal properties which permit adsorption and elution with two chromatographic matrices. It is of considerable interest that separation of chylomicrons from remnants takes place as two discrete elution peaks, i.e., no intermediate forms are seen.

CHYLOYICRONS x-80%

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Fractions (0.75ml) Fig. 1. Separation of chylomicrons from remnants (RI@) by column c~omato~aphy with DEAESephacel (Ph~acia). The salt gradient was determined by conductivity measurements in a separate experiment, but is plotted on all three graphs.

Fig. 1 shows typical c~omato~ap~c profiles of radiolabeled chylomicrons alone, remnants alone and a mixture of chylomicrons plus remnants on a column of DEAE-Sephacel eluted with an NaCl gradient. Recovery of triacylglycerol and radioactivity varied between 80 and 97%. The use of DEAE-Sephacel is inexpensive and has essentially the same effectiveness as the protamine column in separating chylomicrons from remnants. Fig. 2 shows typical chromatographic profiles of radiolabeled chylomicrons alone, remnants alone and a mixture of chylo~crons plus remnants on a column of prota~e-Affi-Gel-10 eluted with an NaCl gradient. The lo-carbon spacer arm is essential to the function of this column, since immobilized protamine (Pierce) did not interact with remnants. Recovery of triacylglycerol and radioactivity varied between 88-95%. When radiolabeled renmants were chromatographed with unlabeled chylomicrons, little or no transfer of radioactivity was observed. The basis of separation of chylomicrons from remnants is the marked increase in surface negativity when recants are formed. In other experiments we have found that fatty acid-labeled chylomicrons contain less than 1% of the label in the free fatty acid fraction, while remnants contain 3-8% of the label in the free fatty acid fraction when analyzed by the technique of Schotz et al. [15] (Itakura, H. and Felts, J.M., unpublished data). Thus, the high negative charge is probably due to surface-oriented free fatty acids, although other negatively charged molecules (like gly~sa~noglyc~s) are also a possibility. In order to test the free fatty acid h~othesis, chromatography was carried out on the protamine column (essentially polyarginine) at a pH of 4.5 using an acetate buffer (0.02 M) in place of Tris. Since fatty acids have a pK, of 4.7-4.8, we selected a pH of 4.5 in order to suppress free fatty acid ionization. Under these conditions remnants did not adhere to the protamine column and eluted in essentially the same volume as chylomicrons. This suggests that surface-oriented free fatty acids provide the negative charge on remnants at pH 8.6. When using these columns for the separation of chylomicrons and remnants in both in vivo and in vitro experiments, it is important that the lipoproteins be isolated by ultracentrifugation before

2.0

CNYLOYICRONS

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Fractions (0.75ml) Fig. 2. Separation of chylomicrons from remnants (RIM) by column chromatography with protamine-Affi-Gel10(Bio-Rad). The salt gradient was determined by conductivity measurements in a separate experiment, but is plotted on all three graphs.

analysis. The presence of albumin or other serum proteins interferes with the efficiency of both columns. In addition, in the presence of large amounts of very low density lipoproteins the negative charge on remnants may be partially negated [13], and therefore quantitative separation of chylomicrons and remnants may not be possible. The mechanism of this charge neutralization is not clear at the present time. Freshly prepared chylomicrons have surface characteristics which do not interact with either chromatographic material, since chylomicrons elute essentially in the void volume in 0.15 NaCl.

However, if stored for a period of days at room temperature, chylomicrons develop a negatively charged component which increases with time. If stored at 0°C for up to 1 week, no negative component develops. The basis for the development of the negative component may be the presence of a small amount of lipase on chylomicron surfaces. Two chromatographic techniques have been described that effect quantitative separation of mixtures of chylomicrons and remnants. These techniques may be of value in studies of the metabolism of chylomicrons in vivo and in vitro. A preliminary abstract of these techniques has been presented [14]. The work was supported by the Medical Research Service of the Veterans Administration and by grant No. AM-21923 from the National Institutes of Health. The expert technical assistance of M.C. Gould, R.A. Gorman and A. Frank is gratefully acknowledged.

References 1 Felts, J.M. and Rudel, L.L. (1980) in Handbook of Experimental Pharmacology, Vol. 41 (Kritchevsky, D., ed.), pp. 151-189, Springer Verlag, Berlin 2 Robinson, D.S. and Harris, P.M. (1959) Q. J. Exp. Physiol. 44, 80-90 3 Felts, J.M. and Mayes, P.A. (1965) Nature 206, 195-196 4 Redgrave, T.B. (1970) J. CIin. Invest. 49, 465-471 5 Noel, S.P., Dolphin, P.J. and Rubinstein, D. (1975) Biothem. Biophys. Res. Commun. 63, 764-772 6 Felts, J.M., Itakura, H. and Crane, R.T. (1975) B&hem. Biophys. Res. Commun. 66, 1467-1475 I Mjos, O.D., Faergemann, O., Hamilton, R.J. and Havel, R.J. (1975) J. Clin. Invest. 56, 603-615 8 Smith, L.C. and Pownall, H.J. (1984) in Lipases (Borgstrom, B. and Brockman, H.L., eds.), pp. 263-306, Elsevier, Amsterdam 9 Bollman, J.L., Cain, J.C. and Grindlay, J.H. (1948) J. Lab. CIin. Med. 33, 1349-1352 10 Berman-Tarcher, A. and Robinson, D.S. (1965) Proc. R. Sot. B. London 162,406-410 11 Kessler, G. and Lederer, H. (1966) in Automation in Analytical Chemistry, Technicon Symposium (Skeggs, L.T., ed.), pp. 341-344, Mediac, Inc., New York 12 Fletcher, M.J. (1968) Clin. Chim. Acta 22, 393-397 13 Felts, J.M., Gould, M.C., Gorman, R.A. and Frank, A. (1983) Circulation 68, III-216 14 Felts, J.M., Gould, M.C., Gorman, R.A. and Frank, A. (1983) Physiologist 26, A-61 15 Schotz, M.C., Garfinkel, A.S., Huebotter, R.J. and Stewart, J.E. (1970) J. Lipid Res. 11, 68-69